It is increasingly clear that pre-mRNA-protein complexes (also known as heterogenous nuclear RNA-protein complexes or hnRNP) play an important role in RNA splicing. We propose that one of the functions of hnRNP in RNA splicing is sequestration of RNA splicing center and stabilization of the quaternary structure in which the splice junctions are in a close proximity during RNA splicing. Certain pre-mRNA structures in which the splice junctions are artificially brought into a close proximity appear to be spliced in vitro in the absence of hnRNP formation and perhaps mimic the conformation of hnRNP involving the splicing reaction center. We believe that careful analysis of various pre-mRNA structures will provide important information on the role of hnRNP in RNA splicing. In this application we will construct various pre-mRNA structures in which the splice junctions are in a close proximity by hybridization of chicken b-globin and mouse metallothionein-I pre-mRNAs with cRNA and also by forming a stem structure with complementary exon sequences. Such pre- mRNAs will be spliced in HeLa nuclear extracts and the spliced products and intermediates will be analyzed by high resolution polyacrylamide gel electrophoresis. In addition, we will investigate binding of hnRNP proteins, assembly of hnRNP, requirement of snRNPs for splicing, and possible sequestration of splicing reaction in the absence of hnRNP formation.